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Restaining cells after cryopreservation - (Mar/05/2008 )

I stained cells with different monoclonal antibodies. Unfortunately after staining the cells, our flow cytometer had broken down. I had the cells pulled and cryopreserved. I wonder would I be able to stain the cells with different monoclonal antibodies again after thawing them later?
Thx smile.gif

-yftan2-

So, you stained cells for flow, and freeze the cell?
my questin is your stained cell still alive? or you fixed?
if cell is still viable you can stain again with different antibody, no problem.
if you fixed the cell, prob not.



QUOTE (yftan2 @ Mar 5 2008, 05:32 PM)
I stained cells with different monoclonal antibodies. Unfortunately after staining the cells, our flow cytometer had broken down. I had the cells pulled and cryopreserved. I wonder would I be able to stain the cells with different monoclonal antibodies again after thawing them later?
Thx smile.gif

-Rnotk-

Yes. I stained the cell for flow cytometry analysis, I freeze them up becoz our flow cytometer broken down.
The cells are still alive, I didn't fix them.
I need to stain them again with the same set of monoclonal antibodies. Just wondering whether these cells can still pick up the monoclonal antibodiyes as their receptors might be taken up by the previous staining.
Thx.

QUOTE (Rnotk @ Mar 7 2008, 02:05 AM)
So, you stained cells for flow, and freeze the cell?
my questin is your stained cell still alive? or you fixed?
if cell is still viable you can stain again with different antibody, no problem.
if you fixed the cell, prob not.



QUOTE (yftan2 @ Mar 5 2008, 05:32 PM)
I stained cells with different monoclonal antibodies. Unfortunately after staining the cells, our flow cytometer had broken down. I had the cells pulled and cryopreserved. I wonder would I be able to stain the cells with different monoclonal antibodies again after thawing them later?
Thx smile.gif

-yftan2-

well, I never done what you did for your cells, so this is just my assumption

if the cell is not fixed and alive, after certain time you staied with antibody, cell will do so called "capping"
capping is basically cell realize that some antibody attached to the protein so cell will internalize the protein with antibody and digest them.

so if capping happen before you freeze, I think your cell is back to original condition (before you stain with antibody).

However, I am not sure the effect of freezing to the expression of the antigen to your antibody,
on the other hands, Freezing decrease expression of certain protein that you want to detect by your antibody, you might get unexpected result.

again, those are just my opinion, since I never freeze stained cell, so I hope it help, but if this is not what you are looking for, than I am sorry sleep.gif




QUOTE (yftan2 @ Mar 6 2008, 07:59 PM)
Yes. I stained the cell for flow cytometry analysis, I freeze them up becoz our flow cytometer broken down.
The cells are still alive, I didn't fix them.
I need to stain them again with the same set of monoclonal antibodies. Just wondering whether these cells can still pick up the monoclonal antibodiyes as their receptors might be taken up by the previous staining.
Thx.

QUOTE (Rnotk @ Mar 7 2008, 02:05 AM)
So, you stained cells for flow, and freeze the cell?
my questin is your stained cell still alive? or you fixed?
if cell is still viable you can stain again with different antibody, no problem.
if you fixed the cell, prob not.



QUOTE (yftan2 @ Mar 5 2008, 05:32 PM)
I stained cells with different monoclonal antibodies. Unfortunately after staining the cells, our flow cytometer had broken down. I had the cells pulled and cryopreserved. I wonder would I be able to stain the cells with different monoclonal antibodies again after thawing them later?
Thx smile.gif


-Rnotk-

thx a lot. smile.gif

-yftan2-

thawing cyropreserved cells does indeed affect the exression of certain cellular antigens (especially on the cell surface). for example, adhesion molecules and chemokine receptors are literally ripped off the cell surface either during the freezing or the thawing step. to overcome this caveat, simply thaw your cells normally and rest them overnight (37 degrees) in media containing a substantial amount of fbs (or your favorite serum).

once you fix your cells in formaldehyde you would think that it would be possible to 'restain' you cells. this is true for some antibody-fluorophore pairs but most don't work.

i have never stained cryopreserved cells then frozen them down to restain (and i don't know anyone who has, but this is certainly interesting). here's what is likely occurring ... your antibodies (and fluorphores) will likely not react too well to thawing from cryopreservation. i would bet that you wouldn't even detect them, but this is solely due to the thawing step, not a so-called 'capping.'

i have never heard of the term 'capping' but internalization of some cell surface receptors does occur when cross-linked with antibody, this is the exact reason to stain cells at 4C. at this temperature no type of cellular endocytosis occurs (including phagocytosis or pinocytosis).

bottom line, you can certainly try to restain your cells. let us know if this experiment works!

-JE UMass IVP-

buzhidao must be lost till now but this topic is interesting and just adding a few lines to this seeing it appear again.

The results of the experiment will be unreliable and it is wrong to believe these data unless you can confirm that the results are correct by comparing with appropriate controls and also seeing if it is reproducible also. If U r working for some experiment and thinking of publishing these data then it is important that the data be obtained from standard protocol that you can mention in the 'methodology'. If U mention all these in the 'methodology', journals might not accept your manuscript unless U can show that Ur data though U did all these are reliable and reproducible. If U don't mention these then it will be faking your data.

It is better to abandon this and start with fresh cells. U must have done the cytometry with cells suspended in FACS Buffer with Na-Azide. Freezing these cells and expecting them to do well is too much to ask I guess - though I have never done these.

-Bungalow Boy-

QUOTE (Bungalow Boy @ Dec 6 2008, 09:10 AM)
buzhidao must be lost till now but this topic is interesting and just adding a few lines to this seeing it appear again.

The results of the experiment will be unreliable and it is wrong to believe these data unless you can confirm that the results are correct by comparing with appropriate controls and also seeing if it is reproducible also.

It is better to abandon this and start with fresh cells. U must have done the cytometry with cells suspended in FACS Buffer with Na-Azide. Freezing these cells and expecting them to do well is too much to ask I guess - though I have never done these.


This is very good point. Certainly you cannot use the data from these expts for anything other than a 'lab meeting' environment. I definitely agree, that for meaningful data, you need to start over. It would be personally interesting to me to see if you could in fact do the expt that was suggested.

Upon further thinking about this issue, I would have to assume that there would have to be some really extreme circumstances for your cytometer to 'break.' Usually most technical problems with a flow cytometer can be fixed in a few days (in the U.S.) assuming you have a service contract. And this is usually enough time for you to fix your cells and flow them when the instrument is up and running again.

-JE UMass IVP-